This report presents evidence for diversity in membrane binding sites between three forms of ankyrin: brain ankyrin, erythrocyte ankyrin, and a variant of erythrocyte ankyrin (protein 2.2) present in circulating human erythrocytes that is missing a regulatory domain. These ankyrins were compared with respect to binding to kidney microsomes and exhibited the following behavior. 1) Brain and erythrocyte ankyrin each bind to distinct sites. 2) Protein 2.2 is an activated ankyrin that binds to all of the sites accessible to both brain and erythrocyte ankyrin and, in addition, associates with its own specialized sites. 3) The specificity of these membrane sites for various ankyrins is not absolute but reflects 2.5-10-fold differences in relative affinities. Further evidence that binding sites of different ankyrins share some common features is that the cytoplasmic domain of the erythrocyte anion transporter associates with all three ankyrins and displaces binding of the ankyrin variants to kidney membranes. The differences between erythrocyte and brain ankyrins in association with kidney membranes are likely to have physiological relevance to kidney because immunologically related isoforms of ankyrin are expressed in this tissue: erythroid ankyrin which is restricted to the basolateral domains of two cell types and a brain-related ankyrin expressed in all cells and present on apical as well as basolateral membrane surfaces. An unanticipated observation was the discovery of a membrane-associated ankyrin protease in kidney that is specific for erythrocyte ankyrin and may selectively activate the erythroid isoform of ankyrin. The variety of binding sites within this group of ankyrin proteins supports the idea that ankyrins are capable of linking a number of different membrane proteins to the spectrin-actin skeleton.